Basement membrane degrading proteases as insect toxins and methods of use for same

ABSTRACT

Novel insect toxins are disclosed for use in pesticidal compositions and methods. According to the invention basement membrane degrading proteases are identified which are capable of acting as insecticidal agents. Polynucleotides are provided which include expression constructs for the expression of the recombinant insecticidal proteases of the invention as recombinant insect pathogens, as well as transgenic plants with a substantial degree of insect resistance.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. Ser. No.09/614,789 filed Jul. 12, 2000 which claims the benefit of U.S.Provisional Application(s) Nos. 60,143,586 filed Jul. 13, 1999, which isincorporated herein, in its entirety, by reference.

ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT

[0002] This invention was made, at least in part, by a grant from theUnited States Department of Agriculture, Agricultural Research ServiceGrant #97-35302-4325; USDA Project No. IOW03483. The Government may havecertain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention relates generally to the field of pest managementfor agriculture. More particularly the invention relates to the use ofcertain proteases as pesticidal toxins, particularly for transgenicinsecticidal protocols. The invention further comprises insecticidalcompositions as well as transgenic techniques including novel expressionconstructs, vectors, methods for the infection of insects and deliveryof toxins, and ultimately for the development of insect resistanttransgenic plants.

BACKGROUND OF THE INVENTION

[0004] One of the goals of agricultural research is to increaseprofitability of agriculture while decreasing its environmental impact.Integrated pest management programs need a diversity of controlstrategies and agents to maximize profits and minimize environmentaldamage. This need comes at a time when there has been a decrease in thediversity of classical pesticides. Although most experts agree thatartificial pest control is needed to maintain our current level ofagricultural productivity, over reliance on non selective pesticides hasled to resistance, destruction of natural enemies, pest resurgence, anddecreased profitability as well as environmental damage.

[0005] Synthetic chemical insecticides are effective for controllingpest insects in a wide variety of agricultural, urban, and public healthsituations. Unfortunately there are significant, often severe, sideeffects associated with the use of these products. Many pest populationshave developed significant resistance to virtually all chemicalinsecticides, requiring higher and higher rates of usage for continuedcontrol. In a number of severe cases, highly resistant pest populationshave developed which cannot be controlled by any available product.Chemical insecticides may also have deleterious effects on non-targetorganisms. Populations of beneficial arthropods, such as predators andparasites, are sometimes more severely affected by chemical applicationsthan the pests themselves. Minor pests, ordinarily held in check bythese beneficial organisms, may become serious pests when their naturalconstraints are removed by the use of chemical insecticides. Thus, newpest problems may be created by attempts to solve established problems.

[0006] Chemical insecticides may also have adverse effects onvertebrates. The use of DDT has been banned in the United States, dueprimarily to the insecticide's great environmental persistence and itsresulting tendency to accumulate in the tissues of predatory birds,thereby disrupting their ability to produce viable eggs. The use ofcarbofuran has been severely restricted due to its avian toxicity, andmany species of fish are known to be quite sensitive to a variety ofinsecticides. A number of insecticides, such as methyl parathion, arealso quite toxic to humans and other mammals, and by accident or misusehave caused a number of human poisonings. Clearly, the field of insectcontrol would benefit greatly from the discovery of insecticides withimproved selectivity for insects and reduced effects on non-targetorganisms.

[0007] The problems described above, along with other concerns includingthe possibility that some insecticides may act as human carcinogens,have created a strong demand for the development of safer methods ofinsect control.

[0008] Insect pathogens have been the objects of much study as potentialpest control agents. Generally, these pathogens are quite selective forinsects and in many cases affect only a few closely related species ofinsects. A number of insect pathogens have been developed as products,including bacteria (e.g., Bacillus thuringiensis and Bacillus popiliae),viruses (e.g., nucleopolyhedroviruses) and protozoa (e.g., themicrosporidian Nosema locustae). These products occupy only a smallfraction of the insecticide market. Although pathogens may ultimatelycause a high level of mortality in pest populations, the insects maytake weeks to die and continue to feed for much of that time. Thus, anunacceptably high level of crop or commodity damage may be inflictedbefore control is achieved. Currently, researchers are actively seekingways to improve the effectiveness of insect pathogens and otherbiological control tools.

[0009] Insecticidal toxins from arthropods have been the objects ofincreasing interest over the past decade. These materials have proveduseful for the detailed study of neural and neuromuscular physiology ininsects. They have also been used to enhance the effectiveness ofcertain insect pathogens. The insecticidal toxin AaIT, from the scorpionAndroctonus australis, has been employed for both purposes. This toxinbelongs to a group of peptides that are lethal to a variety of insectsbut have no detectable effect in humans. Other toxins in A. australisvenom are lethal to mammals but have no effect on insects. Understandingthe molecular basis of this selectivity may lead to the development ofchemical insecticides with reduced effects on mammals and othernon-target organisms.

[0010] A number of transgenic protocols have been employed to helpreduce the environmental impact of non-selective pesticides. A summaryof current protocols follows. One method involves transformation ofplants with plant disease resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant variety can betransformed with a cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistanceto Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. tomato encodes aprotein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2gene for resistance to Pseudomonas syringae).

[0011] The most extensively used heterologous gene for insect resistanceinvolves the Bt endotoxin. A Bacillus thuringiensis protein, aderivative thereof or a synthetic polypeptide modeled thereon. See, forexample, Geiser et al., Gene 48:109 (1986), who disclose the cloning andnucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNAmolecules encoding delta-endotoxin genes can be purchased from AmericanType Culture Collection (Rockville, Md.), for example, under ATCCAccession Nos. 40098, 67136, 31995 and 31998. Other examples include useof a lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes, use of avitamin-binding protein, such as avidin. See PCT application US93/06487the contents of which are hereby incorporated by reference. (Theapplication teaches the use of avidin and avidin homologues aslarvicides against insect pests); and use of an enzyme inhibitor, forexample, a protease inhibitor or an amylase inhibitor. See, for example,Abe et al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of ricecysteine proteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985(1993) (nucleotide sequence of cDNA encoding tobacco proteinaseinhibitor I), and Sumitani et al., Biosci. Biotech. Biochem. 57:1243(1993) (nucleotide sequence of Streptomyces nitrosporeus alpha-amylaseinhibitor).

[0012] Still other recombinant strategies include use of insect-specifichormones or pheromone such as an ecdysteroid and juvenile hormone, avariant thereof, a mimetic based thereon, or an antagonist or agonistthereof. See, for example, the disclosure by Hammock et al., Nature344:458 (1990), of baculovirus expression of cloned juvenile hormoneesterase, an inactivator of juvenile hormone.

[0013] Further techniques include an insect-specific peptide orneuropeptide which, upon expression, disrupts the physiology of theaffected pest. For example, see the disclosures of Regan, J. Biol. Chem.269:9 (1994) (expression cloning yields DNA coding for insect diuretichormone receptor), and Pratt et al., Biochem. Biophys. Res.Comm.163:1243 (1989) (an allostatin is identified in Diplopterapuntata). See also U.S. Pat. No. 5,266,317 to Tomalski et al., whodisclose genes encoding insect-specific, paralytic neurotoxins. Anenzyme involved in the modification, including the post-translationalmodification, of a biologically active molecule; for example, achitinase, whether natural or synthetic has been used to createresistant transgenic plants. See PCT application WO 93/02197 in the nameof Scott et al., which discloses the nucleotide sequence of a callasegene. DNA molecules which contain chitinase-encoding sequences can beobtained, for example, from the ATCC under Accession Nos. 39637 and67152. See also Kramer et al., Insect Biochem. Molec. Biol.23:691(1993), who teach the nucleotide sequence of a cDNA encoding tobaccohornworm chitinase, and Kawalleck et al., Plant Molec. Biol. 21:673(1993), who provide the nucleotide sequence of the parsley ubi4-2polyubiquitin gene.

[0014] A molecule that stimulates signal transduction is yet anotherclass of proteins. For example, see the disclosure by Botella et al.,Plant Molec. Biol. 24:757 (1994), of nucleotide sequences for mung beancalmodulin cDNA clones, and Griess et al., Plant Physiol.104:1467(1994), who provide the nucleotide sequence of a maize calmodulin cDNAclone. A hydrophobic mutant peptide has been used. See PCT applicationWO95/16776 (disclosure of peptide derivatives of Tachyplesin whichinhibit fungal plant pathogens) and PCT application WO95/18855 (teachessynthetic antimicrobial peptides that confer disease resistance), therespective contents of which are hereby incorporated by reference. Amembrane permease, a channel former or a channel blocker has been used.For example, see the disclosure by Jaynes et al., Plant Sci. 89:43(1993), of heterologous expression of a cecropin lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

[0015] Yet another technique includes a viral-invasive protein or acomplex toxin derived therefrom. For example, the accumulation of viralcoat proteins in transformed plant cells imparts resistance to viralinfection and/or disease development effected by the virus from whichthe coat protein gene is derived, as well as by related viruses. SeeBeachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coatprotein-mediated resistance has been conferred upon transformed plantsagainst alfalfa mosaic virus, cucumber mosaic virus, tobacco streakvirus, potato virus X, potato virus Y, tobacco etch virus, tobaccorattle virus and tobacco mosaic virus. Id.

[0016] An insect-specific antibody or an immunotoxin derived therefromhas been used. Thus, an antibody targeted to a critical metabolicfunction in the insect gut would inactivate an affected enzyme, killingthe insect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland, 1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

[0017] Finally, a virus-specific antibody has been shown to conferprotection. See, for example, Tavladoraki et al., Nature 366:469 (1993),who show that transgenic plants expressing recombinant antibody genesare protected from virus attack. As can be seen from the foregoing thereis a continuing need for environmentally safe alternatives to chemicalpesticides.

[0018] It is an object of the present invention to provide geneticallyengineered insect pathogens which express a novel toxin.

[0019] It is yet another object of the invention to provide transgenicplants which express a non specific insect toxin to engineer insectresistant plants.

[0020] It is yet another object to provide expression constructs,vectors, and protocols for providing the insect pathogen and transgenicplants of the invention.

[0021] Other objects of the invention will become apparent from thedetailed description of the invention which follows.

SUMMARY OF THE INVENTION

[0022] According to the invention, applicants have discovered thatproteases which degrade or disrupt basement membranes such asmetalloproteases, collagenases, gelatinases, stromelysins, cysteineproteases, as well as basement degrading proteases from snake venom,invertebrates, fungi, and bacteria are useful as insecticidal toxins.Surprisingly applicants have discovered that the basement membranedegrading proteases themselves act as toxins and may be used asinsecticidal agents with efficacy against a variety of pest species.

[0023] When produced within insect tissues the protease is exported fromthe cells and degrades the basement membrane surrounding the tissues.Basement membranes provide structural support, a filtration function anda surface for cell attachment, migration and differentiation.Degradation of the basement membrane results in rapid death of theinsect.

[0024] According to the invention polynucleotides are provided whichinclude expression constructs for the expression of recombinantinsecticidal proteases in insect pathogens or in transgenic plants. Theexpression constructs may comprise regulatory elements such as promotersand termination signals which are effective in the particular host cellor recipient (pathogen or plant) of the construct.

[0025] For purposes of this application the following terms shall havethe definitions recited herein. Units, prefixes, and symbols may bedenoted in their SI accepted form. Unless otherwise indicated, nucleicacids are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation,respectively. Numeric ranges are inclusive of the numbers defining therange and include each integer within the defined range. Amino acids maybe referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes. The termsdefined below are more fully defined by reference to the specificationas a whole.

[0026] As used herein the term “basement membrane degrading protease”shall include any protease capable of digesting or otherwise disruptingthe basement membrane of a desired pest. This includes but is notlimited to: Mammalian matrix metalloproteases (MMPs) including:Collagenases: (Interstitial collagenase (MMP-1, fibroblast collagenase,EC 3.4.24.7) Collagenase-3 (MMP-13)Neutrophil collagenase (MMP-5, EC3.4.24.34)PMN-type collagenase (MMP-8); Gelatinases(Gelatinase A (MMP-2,72 kDa type IV collagenase, EC 3.4.24.24); Gelatinase B (MMP-9, 92 kDatype IV collagenase, EC 3.4.24.35)) Stromelysins: (Stromelysin-1 (MMP-3,transin, proteoglycanase, EC 3.4.24.17); Stromelysin-2 (MMP-10,transin-2, EC 3.4.24.22), Stromelysin-3 (MMP-11), Matrilysin (MMP-7,pump-1, EC 3.4.24.23); Metalloelastase (MMP-12); membrane-type MMP(MMP-14) Mammalian cysteine proteases, including Cathepsin B (EC3.4.22.1), Cathepsin L (EC 3.4.22.15), Cathepsin N Snake venomproteases, including, Crotalus atrox (Western diamondback rattlesnake)and hemorrhagic metalloproteinases: (Ht-a, Ht-c, Ht-d, and Ht-e)Invertebrate proteases, including: Hypoderma lineatum (fly) collagenase(EC 3.4.21.49), Uca pugilator (crab) collagenolytic endopeptidase (EC3.4.21.32), Fungal proteases, such as Entomophthora collagenase (EC3.4.21.33) Bacterial proteases, including: Clostridium histolyticumcollagenase (EC 3.4.24.03), Streptomyces collagenase, Serratiamarcescens cysteine endopeptidase.

[0027] The term is also intended to include conservatively modifiedvariants and other peptide variants which retain enzymatic activity ofsuch proteases. The nucleotide sequences encoding these enzymes aregenerally known to those of skill in the art and available throughsources such as Genbank. (See fibroblast collagenase, EC3.4.24.7 Genbankaccession number X05231, PMN-type collagenase (MMP-8)Genbank accessionnumber J05556, Gelatinase B (MMP-9, 92 kDa type IV collagenase,EC3.4.24.35 Genbank accession number J05070, Stromelysin-1 (MMP-3,transin, proteoglycanase, EC3.4.24.17 Genbank accession number X05232),Stromelysin-2 (MMP-10, transin-2, EC 3.4.24.22, Genbank accession numberX07820),Matrilysin (MMP-7, pump-1, EC 3.4.24.23 Genbank accession numberX07819), Metalloelastase (MMP-12) Genbank accession number L23808,Cathepsin B (EC 3.4.22.1) Genbank accession number M14221, Cathepsin L(EC 3.4.22.15) Genbank accession number L23808, Crotalus atrox (Westerndiamondback rattlesnake) hemorrhagic metalloproteinases: Ht-a, Ht-c,Ht-d, and Ht-e Accession numbers: Ht-a: U01234, Ht-c: U01236, Ht-d:U01237, Uca pugilator (crab) collagenolytic endopeptidase (EC 3.4.21.32)Genbank accession number U49931 (accession # for the Uca enzyme), andClostridium histolyticum collagenase (EC 3.4.24.03) Genbank accessionnumber D29981. Those of skill in the art will appreciate that otherbasement membrane degrading proteases will be applicable to theteachings herein, or will become available or isolated using no morethan routine experimentation.

[0028] By “amplified” is meant the construction of multiple copies of anucleic acid sequence or multiple copies complementary to the nucleicacid sequence using at least one of the nucleic acid sequences as atemplate. Amplification systems include the polymerase chain reaction(PCR) system, ligase chain reaction (LCR) system, nucleic acid sequencebased amplification (NASBA, Canteen, Mississauga, Ontario), Q-BetaReplicase systems, transcription-based amplification system (TAS), andstrand displacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

[0029] The term “conservatively modified variants” applies to both aminoacid and nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or conservatively modified variants of theamino acid sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein that encodes a polypeptide also, by reference to thegenetic code, describes every possible silent variation of the nucleicacid. One of ordinary skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine; andUGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide of the presentinvention is implicit in each described polypeptide sequence and iswithin the scope of the present invention.

[0030] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Thus, any number of amino acid residues selected from thegroup of integers consisting of from 1 to 15 can be so altered. Thus,for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made.Conservatively modified variants typically provide similar biologicalactivity as the unmodified polypeptide sequence from which they arederived. For example, substrate specificity, enzyme activity, orligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%,80%, or 90% of the native protein for its native substrate. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art.

[0031] The following six groups each contain amino acids that areconservative substitutions for one another:

[0032] 1) Alanine (A), Serine (S), Threonine (T);

[0033] 2) Aspartic acid (D), Glutamic acid (E);

[0034] 3) Asparagine (N), Glutamine (Q);

[0035] 4) Arginine (R), Lysine (K);

[0036] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0037] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also,Creighton (1984) Proteins W.H. Freeman and Company.

[0038] By “encoding” or “encoded”, with respect to a specified nucleicacid, is meant comprising the information for translation into thespecified protein. A nucleic acid encoding a protein may comprisenon-translated sequences (e.g., introns) within translated regions ofthe nucleic acid, or may lack such intervening non-translated sequences(e.g., as in cDNA). The information by which a protein is encoded isspecified by the use of codons. Typically, the amino acid sequence isencoded by the nucleic acid using the “universal” genetic code. However,variants of the universal code, such as are present in some plant,animal, and fungal mitochondria, the bacterium Mycoplasma capricolum, orthe ciliate Macronucleus, may be used when the nucleic acid is expressedtherein.

[0039] When the nucleic acid is prepared or altered synthetically,advantage can be taken of known codon preferences of the intended hostwhere the nucleic acid is to be expressed. For example, although nucleicacid sequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)).Thus, the maize preferred codon for a particular amino acid may bederived from known gene sequences from maize. Maize codon usage for 28genes from maize plants are listed in Table 4 of Murray et al., supra.

[0040] As used herein, “heterologous” in reference to a nucleic acid isa nucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

[0041] By “host cell” is meant a cell which contains a vector andsupports the replication and/or expression of the vector. Host cells maybe prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells or insect cells.

[0042] The term “hybridization complex” includes reference to a duplexnucleic acid structure formed by two single-stranded nucleic acidsequences selectively hybridized with each other.

[0043] The term “introduced” in the context of inserting a nucleic acidinto a cell, means “transfection” or “transformation” or “transduction”and includes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

[0044] The term “isolated” refers to material, such as a nucleic acid ora protein, which is: (1) substantially or essentially free fromcomponents that normally accompany or interact with it as found in itsnaturally occurring environment. The isolated material optionallycomprises material not found with the material in its naturalenvironment; or (2) if the material is in its natural environment, thematerial has been synthetically (non-naturally) altered by deliberatehuman intervention to a composition and/or placed at a location in thecell (e.g., genome or subcellular organelle) not native to a materialfound in that environment. The alteration to yield the syntheticmaterial can be performed on the material within or removed from itsnatural state. For example, a naturally occurring nucleic acid becomesan isolated nucleic acid if it is altered, or if it is transcribed fromDNA which has been altered, by means of human intervention performedwithin the cell from which it originates. See, e.g., Compounds andMethods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S.Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in EukaryoticCells; Zarling et al., PCT/US93/03868. Likewise, a naturally occurringnucleic acid (e.g., a promoter) becomes isolated if it is introduced bynon-naturally occurring means to a locus of the genome not native tothat nucleic acid. Nucleic acids which are “isolated” as defined herein,are also referred to as “heterologous” nucleic acids.

[0045] As used herein, “nucleic acid” or “nucleotide” includes referenceto a deoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

[0046] As used herein “operably linked” includes reference to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame.

[0047] As used herein, the term “plant” can include reference to wholeplants, plant parts or organs (e.g., leaves, stems, roots, etc.), plantcells, seeds and progeny of same. Plant cell, as used herein, furtherincludes, without limitation, cells obtained from or found in: seeds,suspension cultures, embryos, meristematic regions, callus tissue,leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. Plant cells can also be understood to include modifiedcells, such as protoplasts, obtained from the aforementioned tissues.The class of plants which can be used in the methods of the invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants. Particularly preferred plants are agriculturalplants.

[0048] As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof thathave the essential nature of a natural ribonucleotide in that theyhybridize, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallow translation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons as “polynucleotides” as thatterm is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

[0049] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. It will be appreciated, as is wellknown and as noted above, that polypeptides are not entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination,and they may be circular, with or without branching, generally as aresult of post translation events, including natural processing eventand events brought about by human manipulation which do not occurnaturally. Circular, branched and branched circular polypeptides may besynthesized by non-translation natural process and by entirely syntheticmethods, as well.

[0050] As used herein “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria which comprise genes expressed inplant cells such as Agrobacterium or Rhizobium. Examples of promotersunder developmental control include promoters that preferentiallyinitiate transcription in certain tissues, such as leaves, roots, orseeds. Such promoters are referred to as “tissue preferred”. Promoterswhich initiate transcription only in certain tissue are referred to as“tissue specific”. A “cell type” specific promoter primarily drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” or “repressible”promoter is a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light. Tissuespecific, tissue preferred, cell type specific, and inducible promotersconstitute the class of “non-constitutive” promoters. A “constitutive”promoter is a promoter which is active under most environmentalconditions.

[0051] As used herein “recombinant” includes reference to a cell orvector, that has been modified by the introduction of a heterologousnucleic acid or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed,under-expressed or not expressed at all as a result of deliberate humanintervention. The term “recombinant” as used herein does not encompassthe alteration of the cell or vector by naturally occurring events(e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

[0052] As used herein, an “expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

[0053] The term “residue” or “amino acid residue” or “amino acid” isused interchangeably herein to refer to an amino acid that isincorporated into a protein, polypeptide, or peptide (collectively“protein”). The amino acid may be a naturally occurring amino acid and,unless otherwise limited, may encompass non-natural analogs of naturalamino acids that can function in a similar manner as naturally occurringamino acids.

[0054] As used herein, “transgenic plant” includes reference to a plantwhich comprises within its genome a heterologous polynucleotide.Generally, the heterologous polynucleotide is stably integrated withinthe genome such that the polynucleotide is passed on to successivegenerations. The heterologous polynucleotide may be integrated into thegenome alone or as part of a recombinant expression cassette.“Transgenic” is used herein to include any cell, cell line, callus,tissue, plant part or plant, the genotype of which has been altered bythe presence of heterologous nucleic acid including those transgenicsinitially so altered as well as those created by sexual crosses orasexual propagation from the initial transgenic. The term “transgenic”as used herein does not encompass the alteration of the genome(chromosomal or extra-chromosomal) by conventional plant breedingmethods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

[0055] As used herein, “vector” includes reference to a nucleic acidused in transfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

[0056] A “structural gene” is a DNA sequence that is transcribed intomessenger RNA (mRNA) which is then translated into a sequence of aminoacids characteristic of a specific polypeptide.

[0057] The term “expression” refers to biosynthesis of a gene product.Structural gene expression involves transcription of the structural geneinto mRNA and then translation of the MRNA into one or morepolypeptides.

[0058] A “cloning vector” is a DNA molecule such as a plasmid, cosmid,or bacterial phage that has the capability of replicating autonomouslyin a host cell. Cloning vectors typically contain one or a small numberof restriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss ofessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

[0059] An “expression vector” is a DNA molecule comprising a gene thatis expressed in a host cell. Typically, gene expression is placed underthe control of certain regulatory elements including promoters, tissuespecific regulatory elements, and enhancers. Such a gene is said to be“operably linked to” the regulatory elements.

[0060] A “recombinant host” may be any prokaryotic or eukaryotic cellthat contains either a cloning vector or an expression vector. This termalso includes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned genes in the chromosome orgenome of the host cell.

[0061] A “transgenic plant” is a plant having one or more plant cellsthat contain an expression vector. Plant tissue includes differentiatedand undifferentiated tissues or plants, including but not limited toroots, stems, shoots, leaves, pollen, seeds, tumor tissue, and variousforms of cells and culture such as single cells, protoplasm, embryos,and callus tissue. The plant tissue may be in plant or in organ, tissue,or cell culture. These proteins can be used in techniques describedherein as molecular markers in breeding to identify and/or select plantswith improved insect resistance.

[0062] As used herein the term “substantially resistant” refers to thefact that the transformed and transgenic plants of this invention haveresistance to pests that invade, infect, or consume the particular plantspecies when compared to the corresponding non-transgenic ornon-transformed plant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063]FIG. 1. Zymography of proteases expressed by recombinantAutographa californica multicapsid nucleopolyhedroviruses. (A) Caseinzymography of medium from mock-infected High Five cells (mock) and cellsinfected with wild-type AcMNPV (C6) and recombinant AcMNPV expressingrat stromelysin-1 (TV3.STR1, MLF9.STR1). (B) Gelatin zymography ofmedium from mock- and wild-type virus-infected cells and cells infectedwith human gelatinase A-expressing viruses (TV3.GEL, MLF9.GEL).Gelatinase: 0.5 U of purified human gelatinase A (Boehringer Mannheim).Recombinant virus-specific bands are indicated by arrows in bothzymographs. Molecular mass markers (Mr) are indicated in kDa.

[0064]FIG. 2. Detection of proteolytic activity in the medium ofinfected and uninfected High Five cells by azocoll assay. (A) Azocollassays carried out at pH 7.6 of medium from mock-infected cells (mock)and cells infected with wild-type AcMNPV (C6) or recombinant AcMNPVexpressing rat stromelysin-1 (TV3.STR1, MLF9.STR1) or human type IVcollagenase (TV3.GEL, MLF9.GEL). (B) Azocoll assays carried out at pH5.0 of medium from mock-infected cells and cells infected with wild-typeAcMNPV and recombinant AcMNPV expressing S. peregrina cathepsin L(TV3.ScathL, MLF9.ScathL). For each treatment, a duplicate set ofreactions were set up with 1 mM 1,10-phenanthroline (phe) or 10 μMtrans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64). The meansof three replicates with 1 standard deviation are shown.

[0065]FIG. 3. Cuticular melanization of larvae infected withprotease-expressing viruses. Fifth instar H. virescens larvae wereinfected with wild-type AcMNPV-C6 and recombinant AcMNPV expressing ratstromelysin-1 (AcMLF9.STR1) and S. peregrina cathepsin L (AcMLF9.ScathL)from the AcMNPV p6.9 promoter. Larvae were photographed at 4 dpost-infection.

[0066]FIG. 4. Internal melanization of larvae infected withprotease-expressing viruses. Internal anatomy of 5th instar H. virescenslarvae infected with wild-type AcMNPV-C6 (A), AcMLF9.STR1 (B), andAcMLF9.ScathL (C, D) is displayed. Fat body (FB) and midgut (MG) arelabeled. Arrows and arrowheads indicate sites of tissue melanization.Bars: 2 mm (A, B, C), 0.5 mm (D).

DETAILED DESCRIPTION OF THE INVENTION

[0067] Toxins currently used for insecticidal compositions such as theBt toxin act at the gut level. The toxins of the invention provide anovel mechanism of action from within the hemocoel of the insect. Seefor example, National Application No. WO99/US21123, WO2000/15758 Milleret al. These toxins are expected to be active against a variety of pestspecies (unlike Bt toxins) and will provide a valuable strategy forcontrol of pests for which transgenic approaches are not currentlyavailable.

[0068] Various toxins derived from scorpion venom are also currentlyused in insecticidal strategies and have been shown to be active in thehemocoel, but these toxins target the nerves and must accumulate tosufficient levels before resulting in toxicity. The protease toxins ofthe invention are active as soon as they are exported from the insecttissue.

[0069] Basement membranes (BMs) are extracellular layers of protein thatsurround all animal tissues, providing structural support, a filtrationfunction, and a surface for cell attachment, migration, anddifferentiation (Yurchenco, P. D., and O'Rear, J. 1993. Supramolecularorganization of basement membranes. In “Molecular and Cellular Aspectsof Basement Membranes” (D. H. Rohrbach, and R. Timpl, Eds.), pp. 19-47.Academic Press, New York). BMs consist mostly of type IV collagen,laminin, and proteoglycans. The major components of BMs are conservedamong vertebrates and invertebrates (Fessler, J. H., and Fessler, L. I.1989. Drosophila extracellular matrix. Annu. Rev. Cell. Biol. 5,309-339; Pedersen, K. J. 1991. Structure and composition of basementmembranes and of the basal matrix systems in selected invertebrates.Acta Zool. 72, 181-201; Ryerse, J. S. 1998. Basal Laminae. In “Insecta”(F. W. Harrison, and M. Locke, Eds.), Vol. 11A, pp. 3-16. Wiley-Liss,Inc., New York).

[0070] The invention is applicable to insect control in general,including but not limited to the Noctuidae. This family includes some ofthe most destructive agricultural pests including the European cornborer, armyworms and cutworms. The technology may also be applied to avariety of other pests in Iowa including stalk borers, sod web worm, hopvine borer and soybean loopers.

[0071] Polynucleotides encoding the toxins may be introduced into asuitable vector (either live or attenuated). When an insect cell is thehost the vector will traditionally be an insect pathogen such asBacillus thuringiensis, Bacillus popiliae, a nucleopolyhedroviruses(Baculovirus) or microsporidian and applied to the sites of the pest tobe controlled, where the live host will proliferate and be ingested bythe pest according to methods known in the art and exemplified herein(See also, for example U.S. Pat. No. 5,135,867 for such methodsemploying the Bt toxin, or U.S. Pat. No. 5,874,298 for methods employinginsecticidal toxins from wasp) or the toxins may be used to createtransgenic organisms for production and harvesting of the recombinantprotein in bacterial cells, for topically applied insecticides orfinally to create insect resistant transgenic plants expressing thetoxins. For creation of transgenic plants a preferred delivery systemwould be one which will deliver the toxins to the hemocoel of theinsect.

[0072] The invention further comprises expression cassettes comprising apromoter, nucleotide sequence comprising a basement membrane degradingtoxin gene, the expression of which is desired in a plant or microbialcell, and a polyadenylation or stop signal. The expression cassette canbe encompassed in plasmid or viral vectors for transformation of plantprotoplast or microbial cells.

[0073] The invention also encompasses transformed bacterial cells formaintenance and replication of the vector or for production andharvesting of the protease toxins, as well as transformed monocot ordicot cells and ultimately transgenic plants, and breeding materialsdeveloped from the transgenic plants.

[0074] In a preferred embodiment the microbial host is a Baculovirus.Baculoviruses are arthropod-specific pathogens which infect a largenumber of insect pests in the order Lepidoptera (Adams, J. R., andMcClintock, J. T. 1991. Baculoviridae. Nuclear polyhedrosis viruses,part 1: Nuclear polyhedrosis viruses of insects. In “Atlas ofInvertebrate Viruses” (J. R. Adams, and J. R. Bonami, Eds.), pp. 87-204.CRC Press, Boca Raton, Fla.). Because baculoviruses are environmentallybenign and do not infect vertebrates, they have been the subject ofintensive study as possible alternatives or supplements to chemicalinsecticides (Black, B. C., et al., 1997. Commercialization ofbaculoviral insecticides. In “The Baculoviruses” (L. K. Miller, Ed.),pp. 341-387. Plenum Press, New York; Moscardi, F. 1999. Assessment ofthe application of baculoviruses for control of Lepidoptera. Annu. Rev.Entomol. 44, 257-289). Baculoviruses possess double-stranded circularDNA genomes contained within enveloped, rod-shaped virions. Virions areenclosed within a protective crystalline matrix consisting of the viralprotein polyhedrin to form polyhedra. Polyhedra can be applied forinsect control by using conventional insecticidal applicationtechniques. Primary infection of the host insect is initiated whenlarvae ingest polyhedra. The polyhedrin crystalline matrix dissolves inthe alkaline environment of the midgut, releasing virions that infectthe midgut cells. Progeny virus from this primary infection establishsecondary infection of other tissues within the host. After viral geneexpression, DNA replication, and virion assembly, polyhedra are producedin massive quantities in the tissues of the host. When the host diesfrom infection, the cadaver lyses and releases polyhedra into theenvironment to initiate further rounds of infection.

[0075] Recombinant baculoviruses expressing genes that encode a varietyof insect-selective toxins or development-disrupting enzymes andhormones have been shown to kill insects faster and reduce feedingdamage to a greater extent than wild-type baculoviruses (van Beek, N. A.M., and Hughes, P. R. 1998. The response time of insect larvae infectedwith recombinant baculoviruses. J. Invertebr. Pathol. 72, 338-347;Harrison, R. L., and Bonning, B. C. 2000. Use of scorpion neurotoxins toimprove the insecticidal activity of Rachiplusia ou multicapsidnucleopolyhedrovirus. Biol. Control 17, 191-201).

[0076] According to the invention, a recombinant baculovirus Autographacalifornica multicapsid nucleopolyhedroviruses (AcMNPV) that expressesproteases known to digest BM proteins were constructed. Viruses thatexpress rat stromelysin-1 (EC 3.4.24.17, matrix metalloprotease-3),human gelatinase A (EC 3.4.24.24, matrix metalloprotease-2), or acathepsin L (EC 3.4.22.15) from the flesh fly, Sarcophaga peregrinaRobineau-Desvoidy, were produced and tested for reduced survival time ofAcMNPV-infected Heliothis virescens Fabricius. The S. peregrinacathepsin L significantly reduced survival time and feeding damagecaused by infected larvae.

Molecular Biology Techniques

[0077] The following is a non-limiting general overview of Molecularbiology techniques which may be used in performing the methods of theinvention. Those of skill in the art will appreciate that the selectionof suitable expedients such as promoter selection, vector type andtransformation techniques comprises a number of alternatives availableto those of skill in the art and will vary according to the host cellused. The selection of the same for optimization of parameters for thenovel insecticidal strategies herein involves no more than routineexperimentation. A comprehensive review of Baculovirus techniques isdisclosed in (King, L. A., et al., 1992, The Baculovirus ExpressionSystem, London: Chapman & Hall. 229 pp.; O'Reilly, D. R., et al., 1992,Baculovirus Expression Vectors—a Laboratory Manual. New York: Freeman.347 pp.).

[0078] Genetic Engineering of Baculoviruses

[0079] The aim of genetic engineering of baculoviruses for use asinsecticides is to combine the pathogenicity of the virus with theinsecticidal action of a toxin, hormone, or enzyme. Upon infection ofthe insect larva with the recombinant baculovirus, the foreign proteinis expressed. If this protein is toxic to the insect, the insect willdie rapidly from this effect, rather than from the viral infectionitself. The recombinant approach will probably also be used to improveproduction, modify host range, and enhance the utility of various insectviruses as biopesticides. However, the goal of the research reviewedhere is to reduce the time from infection with the recombinant virus todeath of the insect such that feeding damage is below the economicthreshold. This goal necessitates an approximate lethal-time ratio(lethal time of test virus divided by lethal time of wild-type virus)(Bonning et al, 1993, “Lethal ratios: an optimized strategy forpresentation of bioassay data generated from genetically engineeredbaculoviruses, J. Invert. Pathol.62: 196-97) of 0.4-0.5 for control ofinsect pests on many crops. Reduction of the lethal time may alsoenhance farmer or use acceptance of baculovirus insecticides.

[0080] Two major baculovirus-expression systems have been developed forthe production of recombinant proteins for research and clinical use.These are based on the nucleopolyhedrovirus derived from the alfalfalooper, Autographa californica (AcNPV), and a similar virus from thesilkworm, Bombyx mori (BmNPV). The sequences of the entire genomes ofboth AcNPV and BmNPV have now been determined (Ayres, M. D., et al.,1994, “The complete sequence of Autographa californica nuclearpolyhedrosis virus”, Virology 202:586-605; Gomi, S., et al., 1999,“Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus”,J. Gen. Virl. 80:1323-1337). Early engineering work was carried out withBmNPV for high levels of protein production in larvae of B. mori. As B.mori is the only known host for BmNPV, this approach also providedbiological containment for the virus. Most recent work in developing thevirus for insect control has concentrated on AcNPV. A variety ofrecently developed techniques and transfer vectors greatly facilitatethe engineering process (Bishop, D H L, 1992, “Baculovirus expressionvectors”, Sem. Virol. 3:253-64; Davies, A. H., 1994, “Current methodsfor manipulating baculoviruses”, Biotechnology 12:47-50; Luckow, V. A.,1994, “Insect cell expression technology. In Principles and Practice ofProtein Engineering, ed. J L Cleland, C S Craik, pp. 1-27. New York:Wiley & Sons). Protein-expression systems have also been established inother baculoviruses, such as Helicoverpa zea NPV (Corsaro, B. G., etal., 1989, “Transfection of cloned Heliothis zea cell lines with the DNAgenome of the Heliothis zea nuclear polyhedrosis virus”, J. Virol.Methods 25:283-91) and Lymantria dispar NPV (Yu, Z., et al., 1992,“Genetic engineering of a Lymantria dispar nuclear polyhedrosis virusfor expression of foreign genes”, J. Gen. Virol. 73:1509-14; Harrison,R. L., et al., 2000, “Use of scorpion neurotoxins to improve theinsecticidal activity of Rachiplusia ou multicapsidnucleopolyhedrovirus”, Biol. Control, 27(3):292-302). This researchprovides the basis for engineering of these viruses for use as insectpest-control agents in the future.

[0081] The circular genome of AcNPV is approximately 134 kilobase pairs(kb) (Ayres, M. C., et al., 1994, supra). Because of the difficulty ofdirect manipulation of such a large piece of DNA, engineering of abaculovirus is usually carried out in two steps. First, the foreign geneis incorporated into a baculovirus-transfer vector. Most transfervectors used are bacterial plasmid, University of California (pUC),derivatives, which encode an origin of replication for propagation inEscherichia coli and an ampicillin-resistance gene. The pUC fragment isligated to a small segment of DNA taken from the viral genome. Theforeign gene sequence is incorporated into a cloning site downstream ofthe promoter selected to drive expression. For the second step, thetransfer vector is mixed with DNA from the parental virus. Theengineered DNA is incorporated into the virus via homologousrecombination events within the nucleus of cultured insect cells. Unlikegenetic engineering in plants, which results in a rather randomincorporation of new DNA into the genome, the baculovirus system allowsthe precise insertion of foreign DNA without disruption of other genes.No drug-resistance markers are included in the final clone, whicheliminates some of the major objections raised to recombinant organisms(Fox, J. L., 1995, “EPA's first commercial release is still pending”,Biotechnology 13:114-15). Commercial kits and reagents are available forthis work, as well as several excellent manuals (King, L. A., et a.,1992, The Baculovirus Expression System, London: Chapman & Hall. 229pp.; O'Reilly, D. R., et al., 1992, Baculovirus Expression Vectors—aLaboratory Manual. New York: Freeman. 347 pp.). A number of recentlydeveloped alternative approaches for genetic engineering ofbaculoviruses have been reviewed elsewhere (Davies, A. H., 1994, supra).

[0082] Early research involved engineering of these viruses for use asprotein-expression vectors rather than for insect control (Smith, G. E.,et al., 1983, “Production of human-interferon in insect cells infectedwith a baculovirus expression vector”, Mol. Cell. Biol. 3:2156-65). Theapproach involved replacing the gene encoding polyhedrin with theforeign gene of interest. Expression of the foreign gene was driven bythe polyhedrin promoter in a polyhedrin-negative virus. Although theseviruses can be manipulated successfully in cell culture for productionof high levels of foreign protein, they lose any advantage conferred bythe polyhedrin coat that protects the virus from inactivation bydesiccation under field conditions. Replacement of the viral geneencoding the p10 protein (Vlak, J. M., et al., 1990, “Expression of acauliflower mosaic virus gene I using a baculovirus vector based on thep10 gene and a novel selection method”, Virology 178:312-20), which isinvolved in calyx attachment and nuclear lysis, also resulted in reducedviral fitness. The stability of polyhedra produced by p10-negativeviruses is greatly reduced (Williams, G. V., et al., 1989, “Acytopathological investigation of Autographa californica nuclearpolyhedrosis virus p10 gene function using insertion/deletion mutants”,J. Gen. Virol. 70:187-202).

[0083] An alternate approach to replacement of a viral gene with aforeign gene sequence is to duplicate a viral promoter. In thisinstance, none of the viral genes are lost, and promoters of essentialviral genes can be used for expression of foreign proteins. The leveland timing of expression of a particular protein by a recombinantbaculovirus is determined in part by the promoter chosen to drivetranscription of the foreign gene sequence. Currently, the polyhedrinand p10 promoters are used most frequently for expression of recombinantproteins. However, expression under the basic protein promoter washigher in several instances (Bonning, B. C., et al., 1994, “Superiorexpression of juvenile hormone esterase and -galactosidease from thebasic protein promoter of Autographa californica nuclear polyhedrosisvirus compared to the p10 protein and polyhedrin promoters”, J. Gen.Virol. 75:1551-56; Lawrie, A. M., et al., 1993, Baculovirus expressionof urokinase-type plasminogen activator: comparison of late and verylate promoters. Presented at Annu. Meet. Am. Soc. Virol. 12th, Davis CA; Sridhar, P., et al., 1993, “Temporal nature of the promoter and notthe relative strength determines the expression of an extensivelyprocessed protein in a baculovirus system”, FEBS Lett. 315:282-86; Lu,A., et al., 1996, “Signal Sequence and Promoter Effects on the Efficacyof Toxin-Expressing Baculoviruses as Biopesticides” Biological control:theory and applications, 7:320). In the future, the use of earlypromoters, hybrid promoters, and promoters from other species willincrease, particularly with the identification of peptides and proteinsthat disrupt insect biology at lower expression levels.

[0084] As mentioned earlier, the application is not limited toBaculovirus as the insect pathogen, other insect pathogens may also beused such as Bacillus and other pathogens listed herein andtransformation techniques with respect to the same are discussed indetail in the documents incorporated herein by reference.

[0085] The following is a summary of molecular biology techniques withan emphasis on plant transformation.

[0086] Structural Gene

[0087] The insect toxin encoding nucleotide sequence may be used itselfor paired with other structural genes or polynucleotides the expressionof which is desired in a particular cell.

[0088] Promoters

[0089] Promoters selected may be constitutive, inducible, or tissuespecific and may be used in conjunction with naturally occurringflanking coding or transcribed sequences of the desired structuralgene/s or with any other coding or transcribed sequence that is criticalto structural gene formation and/or function.

[0090] It may also be desirable to include some intron sequences in thepromoter constructs since the inclusion of intron sequences in thecoding region may result in enhanced expression and specificity.

[0091] Additionally, regions of one promoter may be joined to regionsfrom a different promoter in order to obtain the desired promoteractivity resulting in a chimeric promoter. Synthetic promoters whichregulate gene expression may also be used.

[0092] The expression system may be further optimized by employingsupplemental elements such as transcription terminators and/or enhancerelements.

[0093] A large number of suitable promoter systems are available. Forexample one constitutive promoter useful for the invention is thecauliflower mosaic virus (CaMV) 35S. It has been shown to be highlyactive in many plant organs and during many stages of development whenintegrated into the genome of transgenic plants including tobacco andpetunia, and has been shown to confer expression in protoplasts of bothdicots and monocots.

[0094] Organ-specific promoters are also well known. For example, the E8promoter is only transcriptionally activated during tomato fruitripening, and can be used to target gene expression in ripening tomatofruit (Deikman and Fischer, EMBO J. (1988) 7:3315; Giovannoni et al.,The Plant Cell (1989) 1:53). The activity of the E8 promoter is notlimited to tomato fruit, but is thought to be compatible with any systemwherein ethylene activates biological processes. Similarly theLipoxegenase (“the LOX gene”) is a fruit specific promoter.

[0095] Root specific promoters include the Cam 35 S promoter disclosedin U.S. patent Ser. No. 391,725 to Coruzzi et al.; the RB7 promoterdisclosed in U.S. Pat. No. 5,459,252 to Conking et al. and the promoterisolated from Brassica napus disclosed in U.S. Pat. No. 5,401,836 toBazczynski et al. which give root specific expression.

[0096] Another important method that can be used to identify cell typespecific promoters that allow even to identification of genes expressedin a single cell is enhancer detection (O'Kane, C., and Gehring, W. J.(1987), “Detection in situ of genomic regulatory elements inDrosophila”, Proc. Natl. Acad. Sci. USA, 84, 9123-9127). This method wasfirst developed in Drosophila and rapidly adapted to mice and plants(Wilson, C., Pearson, R. K., Bellen, H. J., O'Kane, C. J., Grossniklaus,U., and Gehring, W. J. (1989), “P-element-mediated enhancer detection:an efficient method for isolating and characterizing developmentallyregulated genes in Drosophila”, Genes & Dev., 3, 1301-1313; Skarnes, W.C. (1990), “Entrapment vectors: a new tool for mammalian genetics”,Biotechnology, 8, 827-831; Topping, J. F., Wei, W., and Lindsey, K.(1991), “Functional tagging of regulatory elements in the plant genome”,Development, 112, 1009-1019; Sundaresan, V., Springer, P. S., Volpe, T.,Haward, S., Jones, J. D. G., Dean, C., Ma, H., and Martienssen, R. A.,(1995), “Patterns of gene action in plant development revealed byenhancer trap and gene trap transposable elements”, Genes & Dev., 9,1797-1810).

[0097] The promoter used in the method of the invention may be aninducible promoter. An inducible promoter is a promoter that is capableof directly or indirectly activating transcription of a DNA sequence inresponse to an inducer. In the absence of an inducer, the DNA sequencewill not be transcribed. Typically, the protein factor that bindsspecifically to an inducible promoter to activate transcription ispresent in an inactive form which is then directly or indirectlyconverted to the active form by the inducer. The inducer may be achemical agent such as a protein, metabolite (sugar, alcohol etc.), agrowth regulator, herbicide, or a phenolic compound or a physiologicalstress imposed directly by heat, salt, toxic elements etc. or indirectlythrough the action of a pathogen or disease agent such as a virus. Aplant cell containing an inducible promoter may be exposed to an inducerby externally applying the inducer to the cell such as by spraying,watering, heating, or similar methods. Examples of inducible promotersinclude the inducible 70 kd heat shock promoter of D. melanogaster(Freeling, M., Bennet, D. C., Maize ADN 1, Ann. Rev. of Genetics,19:297-323) and the alcohol dehydrogenase promoter which is induced byethanol (Nagao, R. T., et al., Miflin, B. J., Ed. Oxford Surveys ofPlant Molecular and Cell Biology, Vol. 3, p. 384-438, Oxford UniversityPress, Oxford 1986) or the Lex A promoter which is triggered withchemical treatment and is available through Ligand pharmaceuticals. Theinducible promoter may be in an induced state throughout seed formationor at least for a period which corresponds to the transcription of theDNA sequence of the recombinant DNA molecule(s).

[0098] Other Regulatory Elements

[0099] In addition to a promoter sequence, an expression cassette orconstruct should also contain a transcription termination regiondownstream of the structural gene to provide for efficient termination.The termination region or polyadenylation signal may be obtained fromthe same gene as the promoter sequence or may be obtained from differentgenes. Polyadenylation sequences for plant cells include, but are notlimited to the Agrobacterium octopine synthase signal (Gielen et al.,EMBO J. (1984) 3:835-846) or the nopaline synthase signal (Depicker etal., Mol. and Appl. Genet. (1982) 1:561-573).

[0100] Marker Genes

[0101] Recombinant DNA molecules containing any of the DNA sequences andpromoters described herein may additionally contain selection markergenes which encode a selection gene product which confer on a plant cellresistance to a chemical agent or physiological stress, or confers adistinguishable phenotypic characteristic to the cells such that plantcells transformed with the recombinant DNA molecule may be easilyselected using a selective agent. One such selection marker gene isneomycin phosphotransferase (NPT II) which confers resistance tokanamycin and the antibiotic G-418. Cells transformed with thisselection marker gene may be selected for by assaying for the presencein vitro of phosphorylation of kanamycin using techniques described inthe literature or by testing for the presence of the mRNA coding for theNPT II gene by Northern blot analysis in RNA from the tissue of thetransformed plant. Polymerase chain reactions are also used to identifythe presence of a transgene or expression using reverse transcriptasePCR amplification to monitor expression and PCR on genomic DNA. Othercommonly used selection markers include the ampicillin resistance gene,the tetracycline resistance and the hygromycin resistance gene.Transformed plant cells thus selected can be induced to differentiateinto plant structures which will eventually yield whole plants. It is tobe understood that a selection marker gene may also be native to aplant.

[0102] Transformation

[0103] A recombinant DNA molecule may be integrated into the genome of aplant by first introducing a recombinant DNA molecule into a plant cellby any one of a variety of known methods. Preferably the recombinant DNAmolecule(s) are inserted into a suitable vector and the vector is usedto introduce the recombinant DNA molecule into a plant cell.

[0104] The use of Cauliflower Mosaic Virus (CaMV) (Howell, S. H., etal., 1980, Science, 208:1265) and gemini viruses (Goodman, R. M., 1981,J. Gen Virol. 54:9) as vectors has been suggested but by far thegreatest reported successes have been with Agrobacteria sp. (Horsch, R.B., et al. 1985, Science 227:1229-1231).

[0105] Methods for the use of Agrobacterium based transformation systemshave now been described for many different species. Generally strains ofbacteria are used that harbor modified versions of the naturallyoccurring Ti plasmid such that DNA is transferred to the host plantwithout the subsequent formation of tumors. These methods involve theinsertion within the borders of the Ti plasmid the DNA to be insertedinto the plant genome linked to a selection marker gene to facilitateselection of transformed cells. Bacteria and plant tissues are culturedtogether to allow transfer of foreign DNA into plant cells thentransformed plants are regenerated on selection media. Any number ofdifferent organs and tissues can serve as targets from Agrobacteriummediated transformation as described specifically for members of theBrassicaceae. These include thin cell layers (Charest, P. J., et al.,1988, Theor. Appl. Genet. 75:438-444), hypocotyls (DeBlock, M., et al.,1989, Plant Physiol. 91:694-701), leaf discs (Feldman, K. A., and Marks,M. D., 1986, Plant Sci. 47:63-69), stems (Fry J., et al., 1987, PlantCell Repts. 6:321-325), cotyledons (Moloney M. M., et al., 1989, PlantCell Repts. 8:238-242) and embryoids (Neuhaus, G., et al., 1987, Theor.Appl. Genet. 75:30-36), or even whole plants using in vacuuminfiltration and floral dip or floral spraying transformation proceduresavailable in Arabidopsis and Medicago at present but likely applicableto other plants in the hear future. It is understood, however, that itmay be desirable in some crops to choose a different tissue or method oftransformation.

[0106] Other methods that have been employed for introducing recombinantmolecules into plant cells involve mechanical means such as direct DNAuptake, liposomes, electroporation (Guerche, P. et al., 1987, PlantScience 52:111-116) and micro-injection (Neuhaus, G., et al., 1987,Theor. Appl. Genet. 75:30-36). The possibility of using microprojectilesand a gun or other device to force small metal particles coated with DNAinto cells has also received considerable attention (Klein, T. M. etal., 1987, Nature 327:70-73).

[0107] It is often desirable to have the DNA sequence in homozygousstate which may require more than one transformation event to create aparental line, requiring transformation with a first and secondrecombinant DNA molecule both of which encode the same gene product. Itis further contemplated in some of the embodiments of the process of theinvention that a plant cell be transformed with a recombinant DNAmolecule containing at least two DNA sequences or be transformed withmore than one recombinant DNA molecule. The DNA sequences or recombinantDNA molecules in such embodiments may be physically linked, by being inthe same vector, or physically separate on different vectors. A cell maybe simultaneously transformed with more than one vector provided thateach vector has a unique selection marker gene. Alternatively, a cellmay be transformed with more than one vector sequentially allowing anintermediate regeneration step after transformation with the firstvector. Further, it may be possible to perform a sexual cross betweenindividual plants or plant lines containing different DNA sequences orrecombinant DNA molecules preferably the DNA sequences or therecombinant molecules are linked or located on the same chromosome, andthen selecting from the progeny of the cross, plants containing both DNAsequences or recombinant DNA molecules.

[0108] Expression of recombinant DNA molecules containing the DNAsequences and promoters described herein in transformed plant cells maybe monitored using Northern blot techniques and/or Southern blottechniques or PCR-based methods known to those of skill in the art.

[0109] The regenerated plants are transferred to standard soilconditions and cultivated in a conventional manner. After the expressionor inhibition cassette is stably incorporated into regeneratedtransgenic plants, it can be transferred to other plants by sexualcrossing. Any of a number of standard breeding techniques can be used,depending upon the species to be crossed.

[0110] It may be useful to generate a number of individual transformedplants with any recombinant construct in order to recover plants freefrom any position effects. It may also be preferable to select plantsthat contain more than one copy of the introduced recombinant DNAmolecule such that high levels of expression of the recombinant moleculeare obtained.

[0111] As indicated above, it may be desirable to produce plant lineswhich are homozygous for a particular gene. In some species this isaccomplished rather easily by the use of anther culture or isolatedmicrospore culture. This is especially true for the oil seed cropBrassica napus (Keller and Armstrong, Z. flanzenzucht 80:100-108, 1978).By using these techniques, it is possible to produce a haploid line thatcarries the inserted gene and then to double the chromosome numbereither spontaneously or by the use of colchicine. This gives rise to aplant that is homozygous for the inserted gene, which can be easilyassayed for if the inserted gene carries with it a suitable selectionmarker gene for detection of plants carrying that gene. Alternatively,plants may be self-fertilized, leading to the production of a mixture ofseed that consists of, in the simplest case, three types, homozygous(25%), heterozygous (50%) and null (25%) for the inserted gene. Althoughit is relatively easy to score null plants from those that contain thegene, it is possible in practice to score the homozygous fromheterozygous plants by southern blot analysis in which careful attentionis paid to the loading of exactly equivalent amounts of DNA from themixed population, and scoring heterozygotes by the intensity of thesignal from a probe specific for the inserted gene. It is advisable toverify the results of the southern blot analysis by allowing eachindependent transformant to self-fertilize, since additional evidencefor homozygosity can be obtained by the simple fact that if the plantwas homozygous for the inserted gene, all of the subsequent plants fromthe selfed seed will contain the gene, while if the plant washeterozygous for the gene, the generation grown from the selfed seedwill contain null plants. Therefore, with simple selfing one can easilyselect homozygous plant lines that can also be confirmed by southernblot analysis.

[0112] Creation of homozygous parental lines makes possible theproduction of hybrid plants and seeds which will contain a modifiedprotein component. Transgenic homozygous parental lines are maintainedwith each parent containing either the first or second recombinant DNAsequence operably linked to a promoter. Also incorporated in this schemeare the advantages of growing a hybrid crop, including the combining ofmore valuable traits and hybrid vigor.

[0113] The following examples serve to better illustrate the inventiondescribed herein and are not intended to limit the invention in any way.All references cited herein are hereby expressly incorporated to thisdocument in their entirety by reference.

EXAMPLES

[0114] Cells, Viruses, and Insects. The Spodoptera frugiperda Smith Sf9cell line (Vaughn, J. L., et al., 1977. The establishment of two celllines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). InVitro 13, 213-217) was maintained in TNM-FH medium (JRH Biosciences,Lenexa, Kans.) supplemented with 3% fetal bovine serum (Intergen,Purchase, N.Y.), antibiotics (1 U/ml penicillin, 1 μg/ml streptomycin;Sigma, St. Louis, Mo.), and 0.1% Pluronic F-68 (JRH Biosciences).Trichoplusia ni Hübner BTI-TN-5B1-4 (“High Five”; Wickham, T. J., etal., 1992. Screening of insect cell lines for the production ofrecombinant proteins and infectious virus in the baculovirus expressionsystem. Biotechnol. Prog. 8, 391-396) cells were maintained in Ex-Cell405 medium (JRH Biosciences) supplemented with antibiotics.

[0115] The wild-type AcMNPV strain C6 (Possee, R. D. 1986. Cell-surfaceexpression of influenza virus haemagglutinin in insect cells using abaculovirus vector. Virus Res. 5, 43-59) and the recombinant virusesdescribed in this study were propagated in Sf9 cells and titered byplaque assay. The recombinant viruses AcMLF9.AaIT and AcMLF9.LqhIT2express the scorpion toxins AaIT and LqhIT2, respectively, from theAcMNPV p6.9 promoter (Harrison and Bonning, 2000, “Use of scorpionneurotoxins to improve the insecticidal activity of Rachiplusia oumulticapsid nucleopolyhedrovirus”, Biol. Control, 17(2):191-201). Eggsof Heliothis virescens were obtained from the USDA/ARS Southern InsectManagement Research Unit in Stoneville, Miss. Larvae were reared on H.virescens diet obtained from Southland Products (Lake Village, Ariz.) at27° C. and a 14:10 light:dark cycle.

[0116] Construction of Recombinant AcMNPV. Protease genes were clonedinto the transfer vectors pAcMLF9 (Harrison and Bonning, 2000, supra)and pAcP(+)IE1TV3 (Jarvis, D. L., et al., 1996. Immediate earlybaculovirus vectors for foreign gene expression in transformed orinfected cells. Protein Express. Purif. 8, 191-203). These vectorsprovide for expression of inserted genes from the promoters of theAcMNPV p6.9 and ie-1 genes, respectively. Both transfer vectors containan intact polyhedrin gene and provide for the production ofocclusion-positive viruses. Three proteases were selected for insertioninto AcMNPV: (1) Rat “activated” stromelysin-1: The stromelysins are agroup of matrix (zinc) metalloproteases that degrade a variety ofextracellular matrix proteins, including type IV collagen and laminin(Birkedal-Hansen, H. (1995). Proteolytic remodeling of extracellularmatrix. Curr. Op. Cell. Biol. 7, 728-735). Matrix metalloproteases suchas the stromelysins are expressed and secreted as inactive zymogens. Aconserved sequence in the propeptides of matrix metalloproteases,PRCG(V/N)PD, is involved in maintaining the latency of these enzymes. Weobtained a form of the rat stromelysin-1 gene with a mutation in theconserved propeptide sequence (Val92 to Gly) that results in theproduction of a fully active form of the protease (Park, A. J., et al.,1991. Mutational analysis of the transin (rat stromelysin) autoinhibitorregion demonstrates a role for residues surrounding the “cysteineswitch”. J. Biol. Chem. 266, 1584-1590). The coding sequence for thisactivated form of rat stromelysin-1 was isolated as an EcoR I fragmentfrom pMMTV-STR1-V/G (Witty, J. P., et al., 1995. Matrix metalloproteasesare expressed during ductal and alveolar mammary morphogenesis, andmisregulation of stromelysin-1 in transgenic mice induces unscheduledalveolar development. Mol. Biol. Cell 6, 1287-1303) and cloned into theEcoR I site of pAcMLF9 and the Stu I site of pAcP(+)IE1TV3. (2) Humangelatinase A: The gelatinases (also known as type IV collagenases)degrade native and denatured collagens and other extracellular matrixproteins (Birkedal-Hansen, 1995, supra). A proline-to-glycinesubstitution was inserted by site-directed mutagenesis in the conservedmatrix metalloprotease propeptide sequence PRCG(V/N)PD at the second,underlined proline (Pro105) of gelatinase A (Collier, I. E., et al.,1988. H-ras oncogene-transformed human bronchial epithelial cells(TBE-1) secrete a single metalloprotease capable of degrading basementmembrane collagen. J. Biol. Chem. 263, 6579-6587) using the TransformerSite-Directed Mutagenesis kit from Clontech (Palo Alto, Calif.). Theactivated gelatinase A coding sequence was then isolated as a Not I—EcoRI fragment and blunt-end ligated into the StuI site of pAcP(+)IE1 TV3and the BglII site of pAcMLF9. (3) Cathepsin L from Sarcophagaperegrina: The cysteine protease cathepsin L is normally anintracellular enzyme. However, a cathepsin L from the flesh fly, S.peregrina is constitutively secreted from an embryonic S. peregrina cellline and also secreted from S. peregrina imaginal discs in response to20-hydroxyecdysone (Homma, K., et al., 1994. Purification,characterization, and cDNA cloning of procathepsin L from the culturemedium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina(flesh fly), and its involvement in the differentiation of imaginaldiscs. J. Biol. Chem. 269, 15258-15264.). The secretion and activity ofthis enzyme correlates with the eversion of imaginal discs during theirdevelopment into primordial adult leg structures in vitro and theselective degradation of two proteins with apparent molecular weights of200 and 210 kDa (Homma and Natori, 1996). These proteins are located onthe surfaces of imaginal discs and are believed to be BM components. TheS. peregrina cathepsin L coding sequence was isolated as a Vsp Ifragment from plasmid pKYH5 (Homma et al., 1994, supra) and cloned byblunt-end ligation into the Bgl II site of pAcMLF9 and the Stu I site ofpAcP(+)IEITV3.

[0117] The resulting constructs were used to make the occlusion-positiverecombinant viruses listed in Table 1 by co-transfection of Sf9 cellswith linearized BacPAK6 viral DNA (Kitts, P. A., and Possee, R. D. 1993.A method for producing recombinant baculovirus expression vectors athigh frequency. Biotechniques 14, 810-817) using calcium phosphateprecipitation (Summers, M. D., and Smith, G. E. 1987. “A Manual ofMethods for Baculovirus Vectors and Insect Cell Culture Procedures.”.Tex. Agric. Exp. Stn. Bull., 1555). Techniques for selection andplaque-purification of recombinant viruses were as described by Summersand Smith (1987). Recombinant virus clones were checked for correctinsertion of foreign sequences by restriction enzyme analysis andpolymerase chain reaction amplification and sequencing of the regionwhere the protease genes were inserted. TABLE 1 Characteristics ofbaculoviruses used in this study Promoter used Virus Gene Inserted forexpression AcMNPV-C6 None (wild-type) — AcMLF9.AaIT AaIT scorpiontoxin^(a) p6.9 AcMLF9.LqhIT2 LqhIT2 scorpion toxin^(a) p6.9AcIE1TV3.STR1 rat stromelysin-1 ie-1 AcMLF9.STR1 rat stromelysin-1 p6.9AcIE1TV3.GEL human gelatinase A ie-1 AcMLF9.GEL human gelatinase A p6.9AcIE1TV3.ScathL flesh fly cathepsin L ie-1 AcMLF9.ScathL flesh flycathepsin L p6.9

[0118] Characterization of expressed protease activity. High Five cellswere seeded into 35 mm diameter dishes at a density of 3×106 cells/dishand infected with AcMNPV-C6 and recombinant viruses at a multiplicity ofinfection (M.O.I.) of 1. At 72 hours post-infection (h p. i.), mediumfrom the infections was harvested and clarified by low-speedcentrifugation (500×g for five minutes). For some treatments, mediumsamples were concentrated by centrifugation through Centricon-30 units(Millipore, Bedford, Mass.).

[0119] Protease activity was measured either by zymography or by assaywith the chromogenic substrate azocoll (Calbiochem, La Jolla, Calif.).For zymography, samples of medium from cells infected with gelatinaseA-expressing viruses were concentrated 3-fold, and samples from cellsinfected with stromelysin-1-expressing viruses were concentrated10-fold. Medium from mock- and wild-type virus-infected cells wereconcentrated 3-fold (for gelatin zymography) or 10-fold (for caseinzymography). Aliquots (20 μl) of infected cell culture medium were mixedwith equal volumes of 2× non-denaturing protein sample buffer (2% sodiumdodecyl sulfate-172 mM Tris-HCl, pH 6.8-28% glycerol-0.2% bromophenolblue) and incubated for 20 min at 37° C. Samples were electrophoresed on10% polyacrylamide gels that contained either 0.1% gelatin (for analysisof gelatinase A expression) or (for analysis of stromelysin-1expression). After electrophoresis, the gels were incubated in 2.5%Triton X-100 for one h at room temperature. The gels were rinsed twicewith distilled H2O and incubated 48 h at 37° C. in 50 mM Tris, pH 7.6-10mM CaCl₂-200 mM NaCl-50 μM ZnSO₄. The gels were stained with Coomassieblue R-250, and proteases were visualized as clear bands in a bluebackground.

[0120] Azocoll assays were carried out as described by Fisher and Werb(1995). For assays on medium from cells infected with the virusesexpressing the mammalian proteases, all medium samples were concentrated10-fold except for medium from AcMLF9.STR1-infected cells. Samples foranalysis of S. peregrina cathepsin L expression were not concentrated.Aliquots of infected cell medium (2.5 μl for AcMLF9.ScathL-infectedcells, 40 μl for all other samples) were mixed with 200 μl of 2 mg/mlazocoll suspended either in 50 mM Tris, pH 7.6-10 mM CaCl₂-200 mMNaCl-50 μM ZnSO₄ (for detection of stromelysin-1 and gelatinase Aactivity) or in 0.1 M sodium acetate, pH 5.0 (for detection of cathepsinL activity) in a final volume of 240 μl. A control reaction withsufficient dispase (Boehringer Mannheim, Indianapolis, Ind.) to digestall azocoll in the reaction tube was used as a standard. To confirm thatazocoll digestion was due to matrix metalloprotease or cysteine proteaseactivity, reactions were set up with 1 mM 1,10-phenanthroline (Sigma), aselective inhibitor of zinc metalloproteases, or 10 μMtrans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64; Sigma), aspecific inhibitor of cysteine proteases. Three replicate samples weretested for each treatment, and duplicate reactions were set up for eachsample. Reactions were incubated for 17 h at 37° C. Undigested azocollwas pelleted by centrifugation at 2000×g for 10 minutes. Absorbance ofthe supernatant was measured at 520 nm for each reaction, and mg azocolldigested/ml medium was calculated based on the absorbance of the dispasecontrol reaction (Fisher, S. J., and Werb, Z. 1995). The catabolism ofextracellular matrix components. In “Extracellular Matrix: A PracticalApproach” (M. A. Haralson, and J. R. Hassell, Eds.), pp. 261-287. OxfordUniversity Press, New York).

[0121] Polyhedra production and bioassays. To produce polyhedra forbioassays, H. virescens larvae molting from 4th to 5th instar wereplaced individually in ⅝-ounce cups with no diet. After completing themolt to 5th instar, the larvae were infected by allowing them to feedupon a small diet cube contaminated with 105 polyhedra derived frominfected Sf9 cells. Some of the larvae infected with AcMNPV-C6,AcMLF9.STR1, and AcMLF9.ScathL were dissected and photographed.Polyhedra were isolated from cadavers by a standard method (O'Reilly, D.R., et al., 1992. “Baculovirus Expression Vectors: A Laboratory Manual.”Freeman, New York).

[0122] Lethal concentration bioassays were conducted using the dropletfeeding method of Hughes and Wood (1981) with five differentconcentrations of occlusions (0.5, 2.0, 5.0, 10.0, and 20.0×10⁵polyhedra/ml) and 35 neonate larvae/concentration. Mortality was scoredat approximately two weeks post-infection, when surviving larvae in eachtreatment had either pupated or were in the pre-pupal stage.Dose-mortality relationships were analyzed by probit analysis using thePOLO program (Russell, R. M., et al., 1977. POLO: A new computer programfor probit analysis. Bull. Entomol. Soc. Am. 23, 209-213). Comparison ofLC50s was carried out by the lethal dose ratio comparison method ofRobertson and Preisler (1992),“Pesticide Bioassays with Arthropods.” CRCPress, Boca Raton, Fla.

[0123] Survival time bioassays were prepared by droplet feeding with anLC₉₉ dose of virus. The doses of AcMNPV-C6, AcMLF9.AaIT, AcMLF9.LqhIT2,AcIE1TV3.STR1, AcMLF9.STR1, AcIE1TV3.GEL, AcMLF9.GEL, AcIE1TV3.ScathL,and AcMLF9.ScathL used against neonate H. virescens were 1.80, 2.38,4.29, 3.44, 4.29, 4.44, 1.74, 2.6, and 3.37×106 polyhedra/ml,respectively. Mortality was scored every 4-12 h, and larvae were countedas dead when they no longer moved when probed. Median survival times(ST₅₀) and 95% confidence limits were calculated using the Kaplan-MeierEstimator (Kalbfleisch and Prentice, 1980) with survivors excluded fromthe analysis. Comparison of ST₅₀s was carried out using the log-ranktest (Kalbfleisch, J. D., and Prentice, R. L. 1980. “The StatisticalAnalysis of Failure Time Data.” Wiley, N.Y.). In addition to survivaltime bioassays carried out with individual viruses, bioassays also wereconducted with combinations of protease viruses and AcMLF9.LqhIT2. Inthis case, the infections were carried out with an LC₉₉ dose of eachvirus to obtain a final dose of 2×LC₉₉. All bioassays were repeated atleast three times over the course of six months.

[0124] To measure feeding damage caused by virus-infected larvae, H.virescens 2nd instar larvae were starved overnight and weremock-infected or infected with a 5×neonate LC₉₉ dose of AcMNPV-C6,AcMLF9.LqhIT2, and AcMLF9.ScathL by the droplet feeding method. Thesedoses resulted in 100% mortality by the end of the bioassay. Infectedand mock-infected larvae were transferred individually to 35 mm and 60mm diameter dishes, respectively, containing pieces of iceberg lettuceon damp filter paper. Lettuce pieces were replaced every 2 to 3 days.The areas of the lettuce pieces were measured with a LI-COR 3100 areameter (LI-COR Inc., Lincoln, Nebr.) before and after feeding. After sixdays, all virus-infected larvae were dead, and the total area consumedby each larva was determined. Results were analyzed by Kruskal-WallisANOVA followed by Dunn's test. Feeding damage assays were repeated threetimes.

[0125] All recombinant viruses encoding proteases (Table 1) producedextracellular protease activity above levels observed in medium frommock or wild-type virus infections. In casein zymographs, two bandsmigrating between the 45 and 66 kDa markers were observed with mediumfrom cells infected with stromelysin-1 expressing viruses, but not withmedium from wild-type or mock-infected cells (FIG. 1A). This result isconsistent with the observed mobility of stromelysin-1, which normallymigrates as a doublet at 57 and 59 kDa in SDS-PAGE (Galazka, G., et al.,1996. APMA (4-aminophenylmercuric acetate) activation of stromelysin-1involves protein interactions in addition to those with cysteine-75 inthe propeptide. Biochemistry 35, 11221-11227). Other bands migratingbetween the 31 and 45 kDa markers were also observed. Some of thesebands were present in the mock and wild-type virus samples, while otherswere assumed to be proteolytic fragments derived from stromelysin-1.Gelatin zymographs showed a band co-migrating with the 66 kDa marker andwith purified human gelatinase A in medium from gelatinase A-expressingviruses, but not in the mock-infected and wild-type virus-infectedmedium (FIG. 1B). Fragments of purified human gelatinase A resultingfrom autodegradation were also visible. For both stromelysin-1 andgelatinase A, viruses utilizing the p6.9 promoter to drive expressionproduced more protease than viruses utilizing the ie-1 promoter. The S.peregrina cathepsin L was not detected by zymography under any of theconditions tested.

[0126] In azocoll assays, proteolytic activity was detected in thesupernatants from cells infected with the stromelysin- andgelatinase-expressing viruses (FIG. 2A). This activity was inhibited by1,10-phenanthroline, indicating that it was due to a zincmetalloprotease. As with the zymography experiments, less activity waspresent in medium from cells infected with viruses that utilized theie-1 promoter to drive protease expression than that obtained with thep6.9 promoter. In medium from cells infected with the cathepsinL-expressing viruses, optimal collagenolytic activity was observed at pH5.0, with less activity at pH 4.0 and no activity at pH 6.0 (data notshown). Homma et al. (1994), supra, reported that the optimal activityof purified S. peregrina cathepsin L was pH 3.0-4.0. With azocoll assayscarried out at pH 5.0, a substantial quantity of E-64-inhibitableprotease activity was detected in the medium of mock-infected cells(FIG. 2B). Less activity was present in AcMNPV-C6-infected cell medium.The medium of cells infected with AcMLF9.ScathL contained approximatelyten-fold more E-64-inhibitable protease activity than that ofwild-type-infected cells. Much less activity was present in the mediumof AcIE1TV3.ScathL-infected cells, although this virus still producedtwice as much activity as wild-type virus-infected cells.

[0127] Expression of the three proteases was also detected in thehemolymph of 5th instar H. virescens larvae infected with AcMLF9.STR1,AcMLF9.GEL, and AcMLF9.ScathL by either zymography or azocoll assay(data not shown). Titers of budded virus in the medium of Sf9 cellsinfected with recombinant viruses were indistinguishable from those ofAcMNPV-C6, and no obvious reduction in polyhedra production was observedin High Five cells infected with the protease-expressing viruses (datanot shown). These observations indicate that the budded and occludedforms of AcMNPV were not degraded by the recombinant proteases.

[0128] Fifth-instar H. virescens larvae infected with AcMLF9.ScathLexhibited extensive cuticular melanization by 4 days p. i., oftenoccurring prior to death of the insect (FIG. 3). No wild-typevirus-infected larvae displayed cuticular melanization at this time.Larvae infected with AcMLF9.STR1 (FIG. 3) and AcIE1TV3.ScathL (notshown) also exhibited cuticular melanization, but to a lesser extentthan that seen with AcMLF9.ScathL-infected larvae. Examination of theinternal anatomy of AcMLF9.ScathL-infected 5th instar larvae revealed avariable degree of melanization of tissues (FIG. 4). No internalmelanization was observed with AcMNPV-C6- or AcMLF9.STR1-infectedlarvae. No melanization of hemolymph was observed in larvae infectedwith any of the viruses, but a considerable amount of fragmentation oftissues was observed in approximately half the larvae infected withAcMLF9.ScathL (not shown).

[0129] Lethal concentration bioassays did not reveal any statisticallysignificant differences among the LC50 values for AcMNPV-C6 and therecombinant viruses (Table 2). TABLE 2 Dose-Mortality Response ofNeonate Larvae Infected with Wild-Type and Recombinant Autographacalifornica nucleopolyhedroviruses^(a) Slope Virus LC₅₀ ^(b) × 10⁵ (95%CL) (±SE) Heterogeneity AcMNPV-C6 0.77a (0.46-1.16) 1.29 0.91 (±.194)AcIE1TV3.ScathL 1.19a (0.82-1.65) 1.74 0.58 (±.224) AcMLF9.ScathL 0.69a(0.43-1.01) 1.51 0.94 (±.217) AcMNPV-C6 0.89a (0.54-1.32) 1.85 0.26(±.282) AcIE1TV3.STR1 0.89a (0.45-1.38) 1.70 0.93 (±.302) AcMLF9.STR10.54a (0.31-0.81) 1.79 0.38 (±.296) AcMNPV-C6 0.70a (0.27-1.33) 1.791.27 (±.257) AcIE1TV3.GEL 0.65a (0.38-0.97) 1.43 0.51 (±.221) AcMLF9.GEL0.58a (0.23-1.08) 1.83 1.23 (±.264)

[0130] However, the median survival time of larvae infected withAcMLF9.ScathL was significantly lower than those infected with AcMNPV-C6and the toxin-expressing viruses AcMLF9.AaIT and AcMLF9.LqhIT2 (Table3). AcMLF9.GEL killed larvae slightly but significantly faster thanAcMNPV-C6 in two out of three trials. No significant reduction insurvival time was observed with larvae infected with any of the otherprotease-expressing viruses, including AcIE1TV3.ScathL. TABLE 3Time-Mortality Response of Neonate Larvae Infected with Wild-Type andRecombinant Autographa californica nucleopolyhedroviruses Viruses ST₅₀(h p. i.) 95% CL Infection with a single virus^(a) AcMNPV-C6 98.0 a91.0-103.5 AcMLF9.AaIT 73.5 b 66.5-78.5 AcMLF9.LqhIT2 66.0 c 59.5-66.0AcIE1TV3.STR1 95.0 a 88.0-100.5 AcMLF9.STR1 99.5 a 94.0-111.0AcIE1TV3.GEL 94.0 ad 87.0-94.0 AcMLF9.GEL 86.5 d 86.5-94.0AcIE1TV3.ScathL 92.5 a 89.0-96.0 AcMLF9.ScathL 48.0 e 48.0-48.0 Mixedinfections^(b) AcMNPV-C6 (2X LC₉₉ dose) 97.5 a 90.0-97.5 AcMLF9.LqhIT2 +C6 62.5 b 59.0-66.0 AcMLF9.ScathL + C6 34.0 c 34.0-49.0 AcIE1TV3.STR1 +Lq 64.0 b 57.5-64.0 AcMLF9.STR1 + Lq 64.0 b 57.5-64.0 AcIE1TV3.GEL + Lq63.0 b 56.5-63.0 AcMLF9.GEL + Lq 63.0 b 56.0-69.5 AcIE1TV3.ScathL + Lq64.5 b 57.5-64.5 AcMLF9.ScathL + Lq 47.5 c 47.5-52.5

[0131] To determine if an additive or synergistic effect on survivaltime could be achieved by simultaneous infection with viruses expressinga BM-degrading protease and a scorpion toxin, survival time bioassayswere set up with larvae infected with an LC₉₉ dose of each kind ofvirus. Regardless of the viruses used in each treatment, larvae in thesebioassays died with an ST₅₀ equivalent to that achieved in dualinfections with either AcMLF9.ScathL or AcMLF9.LqhIT2 together withAcMNPV-C6 (Table 3). These data indicate that no additive or synergisticeffect on survival time was obtained with any combination of protease-and toxin-expressing viruses.

[0132] To measure the effect of Sarcophaga cathepsin L expression onfeeding damage caused by virus-infected larvae, the amount of lettuceconsumed by 2nd instar H. virescens infected with AcMNPV-C6,AcMLF9.LqhIT2, and AcMLF9.ScathL was measured and compared. Wild-typevirus-infected larvae consumed significantly more lettuce than larvaeinfected with either AcMLF9.ScathL or AcMLF9.LqhIT2 (Table 4). Nosignificant difference in the leaf area consumed was detected withlarvae infected with AcMLF9.LqhIT2 or AcMLF9.ScathL. TABLE 4 Amount oflettuce consumed by 2nd instar H. virescens infected with wild-type andrecombinant Autographa californica nucleopolyhedroviruses Treatment nMedian area^(a), cm² 25%-75% Mock 30 29.27 a 24.886-32.964 AcMNPV-C6 30 5.21 b  3.383-6.53 AcMLF9.LqhIT2 30  0.91 c  0.515-1.271 AcMLF9.ScathL30  1.1 c  0.858-1.608

Discussion

[0133] Applicant's original hypothesis was that expression ofBM-degrading proteases would hasten the death of baculovirus-infectedlarvae by accelerating the spread of secondary infection to the host'stissues. Of the protease-expressing AcMNPV that were constructed andtested, AcMLF9.ScathL (expressing S. peregrina cathepsin L from the p6.9promoter) killed H. virescens larvae faster than both wild-type AcMNPVand AcMNPV that expressed scorpion toxins. Although S. peregrinacathepsin L appears to be involved in digesting basement membraneproteins in S. peregrina, the mechanism by which it reduces the survivaltime of infected H. virescens remains to be determined. The melanizationof larvae infected with AcMLF9.ScathL suggests that the reduction insurvival time may be connected to the inappropriate activation ofprophenoloxidase, which is the key enzyme involved in the formation ofmelanin during wound healing and the immune response in insects. S.peregrina cathepsin L may activate the phenoloxidase system directly byeither activating the serine protease cascade that leads to theconversion of inactive prophenoloxidase to its active form or bydirectly activating prophenoloxidase itself. Alternatively, themelanization observed may be a consequence of damage to the basementmembranes by the recombinant cathepsin L. In the fruit fly Drosophilamelanogaster Meigen, tissues with damaged or missing basement membranesundergo melanotic encapsulation (Rizki, R. M., and Rizki, T. M. 1980.Hemocyte responses to implanted tissues in Drosophila melanogasterlarvae. Roux's Archives of Developmental Biology 189, 207-213; Rizki, etal., 1983. Drosophila larval fat body surfaces: Changes in transplantcompatibility during development. Roux's Arch. Dev. Biol. 192, 1-7). Thetissue fragmentation observed in AcMLF9.ScathL-infected larvae is alsoconsistent with disruption of the BM. Apart from facilitating the spreadof infection to other tissues, damage to BMs may itself result in thedeath of the insect, possibly by deregulating the ionic and moleculartraffic between the hemocoel and tissues.

[0134] Although AcMLF9.ScathL killed insects rapidly, AcIE1TV3.ScathL, avirus that expresses the same protease from the ie-1 promoter, did notexhibit any improvement in speed of kill. The AcMNPV ie-1 promoter isweaker than the late p6.9 promoter, but it is activated immediatelyafter infection. We hypothesized that early expression and secretion ofproteases may result in sufficient BM solubilization or perforation toallow the first progeny virus emerging from infected midgut cells toimmediately penetrate through the BMs surrounding the midgut sheath,enter the hemocoel of the host, and infect hemocytes. Also, Jarvis, D.L., et al., 1996. Immediate early baculovirus vectors for foreign geneexpression in transformed or infected cells. Protein Express. Purif. 8,191-203, demonstrated that ie-1 promoter-based expression vectors canproduce as much or more of a biologically active secretory pathwayprotein than conventional baculovirus expression vectors that rely onthe polyhedrin (polh) promoter. However, for all three proteases that weexpressed in AcMNPV, the p6.9 promoter appeared to drive significantlyhigher levels of expression and secretion. It is likely that AcIE1TV3.ScathL simply did not produce sufficient cathepsin L to have an effecton the infected host.

[0135] With the exception of a minor reduction in survival time withAcMLF9.GEL, viruses expressing the mammalian proteases did not exhibitany discernible improvement in insecticidal properties. This result maybe because the protease expressed and secreted by these viruses was atrelatively low levels (compare FIG. 2A to FIG. 2B). Alternatively, themammalian proteases may have failed to bind and digest insect BMproteins. The mammalian proteases also may not have been active withinH. virescens larvae. S. peregrina cathepsin L is normally active at anacid pH. The pH between cells and the surrounding BMs may besufficiently low for S. peregrina cathepsin L activity but may inhibitthe two mammalian proteases.

[0136] If S. peregrina cathepsin L expressed from infected host cellsdamages the BM and facilitates secondary infection, then in a dualinfection of larvae with AcMLF9.ScathL and another virus, both virusesmay be able to disseminate within the host more rapidly and establish amore widespread infection of other tissues. If the second virus encodesa toxin, larger quantities of that toxin may be expressed sooner afterinfection, which may further reduce host survival time. Hence, one wouldexpect to see a synergistic or additive effect on host survival timearising from a dual infection with a protease-expressing virus and atoxin-expressing virus. We did not see any such effect in dualinfections with AcMLF9.ScathL and AcMLF9.LqhIT2. The site of action forscorpion toxins is the voltage-gated sodium channels of nervous tissue.Herrmann, R., et al., 1990. The tolerance of lepidopterous larvae to aninsect selective neurotoxin. Insect Biochem. 20, 625-637 reported thatthe intact ventral nerve cord and motor nerves of Spodoptera littoralisBoisduval were not labeled by [125I]AaIT injected into larvae, whiledesheathed neuronal tissue was specifically labeled. The authorsspeculated that the coverage of nerves with glial cells may present animpermeable barrier to scorpion toxins. By infecting and migratingthrough the tracheae, baculoviruses may be capable of bypassing theglial cell coverage and expressing scorpion toxins in sites where thetoxins have greater access to the nerves. Hence, simply increasing theconcentration of toxin within the hemocoel by enhancing systemicinfection via BM disruption may not result in increased access of toxinsto sodium channels. Dual infections with AcMLF9.ScathL and a virusexpressing another class of insecticidal agent (such as mutated juvenilehormone esterase, EC 3.1.1.1; Bonning, B. C., et al., 1997. Disruptionof lysosomal targeting is associated with insecticidal potency ofjuvenile hormone esterase. Proc. Natl. Acad. Sci. USA 94, 6007-6012;Bonning, B. C., et al., 1999. Insecticidal efficacy of a recombinantbaculovirus expressing JHE-KK, a modified juvenile hormone esterase. J.Invertebr. Pathol. 73, 234-236) may yield the synergistic or additivereduction in survival time that we failed to see with dual infectionsinvolving expression of a scorpion toxin.

[0137] The use of viruses expressing proteases will be less likely toprovoke public anxiety than viruses expressing scorpion toxins.Proteases that target only insect proteins would provide an additionallevel of safety. Open reading frames (ORFs) with sequence similarity tomammalian matrix metalloproteases recently have been reported in thegenomes of a granulovirus (Hayakawa, T., et al., 1999. Sequence analysisof the Xestia c-nigrum granulovirus genome. Virology 262, 277-297) andan entomopoxvirus (Afonso, C. L., et al., 1999. The genome of Melanoplussanguinipes entomopoxvirus. J. Virol. 73, 533-552). It is unclear whatfunction the polypeptides specified by these ORFs may have in thebaculovirus or entomopoxvirus life cycle, although they may acceleratesystemic infection by destroying the host BM. Insect- or insectvirus-derived proteases may augment insecticidal activity ofbaculoviruses to a larger extent than proteases derived from otherorganisms.

What is claimed is:
 1. A method of imparting substantial insectresistance to a plant comprising: introducing to said plant apolynucleotide comprising: a nucleotide sequence which encodes uponexpression a basement membrane degrading protease insect toxin, saidnucleotide sequence operably linked to regulatory elements capable ofdirecting expression thereof in a host cell.
 2. The method of claim 1wherein said host cell is a plant cell.
 3. The method of claim 1 whereinsaid polynucleotide is a vector.
 4. The method of claim 3 wherein saidvector is a plasmid vector.
 5. The method of claim 3 wherein said vectoris a viral vector.
 6. The method of claim 5 wherein said viral vector iscapable of transforming a plant cell.
 7. The method of claim 6 whereinsaid vector is a Agrobacterium based vector.
 8. The method of claim 3wherein said vector is capable of transforming an insect cell.
 9. Themethod of claim 8 wherein said vector is an infectious baculovirus basedvector.
 10. The method of claim 1 wherein said step of introducingcomprises: topically administering said vector to plants.
 11. The methodof claim 1 wherein said step of introducing further comprises:transforming a plant cell with said vector.
 12. The method of claim 1wherein said protease is selected from the group consisting of: ametalloproteinase, a collagenase, a gelatinase, a stromelysin, acysteine protease, and basement membrane degrading proteases from snakevenom, invertebrates, fungi, and bacteria.
 13. The method of claim 12wherein said protease is selected from the group consisting of:interstitial collagenase, fibroblast collagenase, Collagenase-3Neutrophil collagenase, PMN-type collagenase, Gelatinase A, (72 kDa typeIV collagenase), Gelatinase B (92 kDa type IV collagenase),Stromelysin-1, Stromelysin-2, Stromelysin-3, Matrilysin,Metalloelastase, Cathepsin B, Cathepsin L, Cathepsin N, Crotalus atrox,hemorrhagic metalloproteinases: (Ht-a, Ht-c, Ht-d, and Ht-e), Hypodermalineatum (fly) collagenase, Uca pugilator (crab) collagenolyticendopeptidase, Entomophthora collagenase, Clostridium histolyticumcollagenase, Streptomyces collagenase, and Serratia marcescens cysteineendopeptidase.
 14. The method of claim 12 wherein said protease is acathepsin L protease.
 15. The method of claim 14 wherein said cathepsinL protease is from the facefly Sarcophaga peregrina.
 16. An insecticidalcomposition comprising: a recombinant basement membrane degradingprotease protein and a carrier.
 17. The composition of claim 1 whereinsaid protease is selected from the group consisting of: ametalloprotease, a collagenase, a gelatinase, a stromelysin, a cysteineprotease, and basement membrane degrading proteases from snake venom,invertebrates, fungi, and bacteria.
 18. The composition of claim 2wherein said protease is selected from the group consisting of:interstitial collagenase, fibroblast collagenase, Collagenase-3Neutrophil collagenase, PMN-type collagenase, Gelatinase A, (72 kDa typeIV collagenase), Gelatinase B (92 kDa type IV collagenase),Stromelysin-1, Stromelysin-2, Stromelysin-3, Matrilysin,Metalloelastase, Cathepsin B, Cathepsin L, Cathepsin N, Crotalus atrox,hemorrhagic metalloproteinases: (Ht-a, Ht-c, Ht-d, and Ht-e), Hypodermalineatum (fly) collagenase, Uca pugilator (crab) collagenolyticendopeptidase, Entomophthora collagenase, Clostridium histolyticumcollagenase, Streptomyces collagenase, and Serratia marcescens cysteineendopeptidase.
 19. The composition of claim 17 wherein said protease isa cathepsin L protease.
 20. The composition of claim 19 wherein saidcathepsin L protease is from the facefly Sarcophaga peregrina.
 21. Anexpression construct comprising: a gene which encodes a basementmembrane degrading protease operably linked to a promoter, said promotercapable of directing expression in an insect cell.
 22. The expressionconstruct of claim 21 wherein said promoter is selected from the groupconsisting of: p6.9 and ie-1.
 23. The expression construct of claim 21wherein said protease is selected from the group consisting of: ametalloprotease, a collagenase, a gelatinase, a stromelysin, a cysteineprotease, and basement membrane degrading proteases from snake venom,invertebrates, fungi, and bacteria.
 24. The expression construct ofclaim 23 wherein said protease is selected from the group consisting of:interstitial collagenase, fibroblast collagenase, Collagenase-3Neutrophil collagenase, PMN-type collagenase, Gelatinase A, (72 kDa typeIV collagenase), Gelatinase B (92 kDa type IV collagenase),Stromelysin-1, Stromelysin-2, Stromelysin-3, Matrilysin,Metalloelastase, Cathepsin B, Cathepsin L, Cathepsin N, Crotalus atrox,hemorrhagic metalloproteinases: (Ht-a, Ht-c, Ht-d, and Ht-e), Hypodermalineatum (fly) collagenase, Uca pugilator (crab) collagenolyticendopeptidase, Entomophthora collagenase, Clostridium histolyticumcollagenase, Streptomyces collagenase, and Serratia marcescens cysteineendopeptidase.
 25. The expression construct of claim 24 wherein saidprotease is a cathepsin L protease.
 26. The expression construct ofclaim 25 wherein said cathepsin L protease is from the faceflySarcophaga peregrina.
 27. A vector comprising the expression constructof claim
 21. 28. The vector of claim 27 wherein said vector is a viralvector.
 29. The vector of claim 28 wherein said vector is capable ofinfecting an insect cell.
 30. The vector of claim 27 wherein said vectoris selected form the group consisting of: bacteria (e.g., Bacillusthuringiensis and Bacillus popiliae), viruses (e.g.,nucleopolyhedroviruses) and protozoa (e.g., the microsporidian Nosemalocustae).
 31. The vector of claim 30 wherein said vector isAcIE1TV3.STR1, AcMLF9.STR1, AcIE1TV3.GEL, AcMLF9.GEL, AcIE1TV3.ScathL,and AcMLF9.ScathL.
 32. A host cell comprising the vector of claim 27.33. The host cell of claim 32 wherein said host cell is a plant cell.34. A transgenic plant which is substantially resistant to insectinfestation comprising the vector of claim
 27. 35. Seed of the plant ofclaim 34.